Molekulare Analyse von Blockierungsmechanismen AMPAerger Glutamatrezeptoren: Entwicklung neuer Therapiestrategien bei neurodegenerativen Erkrankungen

Zusammenfassung

Durch die Anwendung moderner elektrophysiologischer Verfahren, im Speziellen der „patch-clamp”-Technik in Kombination mit einem Verfahren des ultraschnellen Lösungswechsels an Membranflecken, das die experimentelle Untersuchung schneller postsynaptischer Prozesse erlaubt, wurden an rekombinanten AMPA-Typ-Glutamatrezeptoren in vitro pharmakologische Mechanismen auf molekularer Ebene untersucht. Wir konnten mit diesen Untersuchungen die Blockierungsmechanismen verschiedener Substanzklassen, Pyrazinen und Adamantanen, an verschiedenen rekombinanten AMPA-Typ-Glutamatrezeptoren am einzelnen Kanalprotein nachweisen. Es zeigte sich dass Pyrazinderivate einen reinen kompetitiven Block und Adamantane einen kombinierten Offenkanalblock und kompetitiven Block am Rezeptor verursachen. Durch die genaue Kenntnis und Analyse der molekularen Mechanismen der Rezeptorblockierung könnten zukünftig neue effektivere Therapiestrategien bei Prozessen der akuten oder chronischen Neurodegeneration entwickelt werden.

Abstract

By means of modern electrophysiological processes, in particular the „patch clamp” technique in combination with a process for the ultrarapid change of solvent at a membrane patch, which makes the experimental investigation of rapid, post-synaptic processes possible, the in vitro pharamcological mechanisms of recombinant AMPA-type glutamate receptors were examined at the molecular level. With these experiments we were able to detect the blocking mechanisms of various classes of compounds such as pyrazines and adamantanes at the various recombinant AMPA-type glutamate receptors on individual channel proteins. We found that pyrazine derivatives exert a purely competitive blockage at the receptors while adamantanes effect a combined open channel blockade and a competitive blockage at the receprtors. With the exact knowledge and analysis of the molecular mechanisms of receptor blocking it should be possible to develop new, effective therapeutic strategies against processes of acute or chronic neurodegeneration.

AMPA receptor - patchclamp - block mechanism

Literatur

• 1 Doble A. The role of excitotoxicity in neurodegenerative disease: implications for therapy.  Pharmacol Ther. 1999;  81 163-221
• 2 Dingledine R, Borges K, Bowie D, Traynelis S F. The glutamate receptor ion channels.  Pharmacol Rev. 1999;  51 7-61
• 3 Weiss J, Sensi S. Ca²⁺-Zn²⁺ permeable AMPA or kainate receptor channels: possible key factors in selective neurodegeneration.  TINS. 2000;  23 365-371
• 4 Vandenberghe W, Robberecht W, Brorson J R. AMPA receptor calcium permeability, GluR2 expression and selective motoneuron vulnerability.  J Neurosci. 2000;  20 123-132
• 5 Urushitani M, Nakamizo T, Inoue R, Sawada H, Kihara T, Honda K, Akaike A, Simohama S. NMDA receptor mediated mitochondrial Ca²⁺ overload in acute excitotoxic motor neuron death: a mechanism distinct from chronic neurotoxicity after Ca²⁺ influx.  J Neurosci Res. 2001;  63 377-387
• 6 Boulter J, Hollmann M, O'Shea-Greenfield A, Hartley M, Deneris E, Maron C, Heinemann S. Molecular cloning and functional expression of glutamate receptor subunit genes.  Science. 1990;  249 1033-1037
• 7 Keinänen K, Wisden W, Sommer B, Werner P, Herb A, Verdoorn T A, Sakmann B, Seeburg P H. A family of AMPA-selective glutamate receptors.  Science. 1990;  249 556-560
• 8 Sommer B, Keinänen K, Verdoorn T A, Wisden W, Burnashev N, Herb A, Köhler M, Takagi T, Sakmann B, Seeburg P H. Flip and flop: a cell-specific functional switch in glutamate-operated channels of the CNS.  Science. 1991;  249 1580-1585
• 9 Burnashev N, Monyer H, Seeburg P H, Sakmann B. Divalent ion permeability of AMPA receptor channels is dominated by the edited form of a single subunit.  Neuron. 1992;  8 189-198
• 10 Lomeli H, Moosbacher J, Melcher T, Höger T, Geiger J RP, Kuner T, Monyer H, Higuchi M, Bach A, Seeburg P H. Control of kinetic properties of AMPAreceptor channels by nuclear RNA editing.  Science. 1994;  266 1709-1713
• 11 Colquhoun D, Jonas P, Sakmann B. Action of brief pulses of glutamate on AMPA/kainate receptor in patches from different neurones of rat hippocampal slices.  J Physiol. 1992;  458 261-287
• 12 Jahn K, Bufler J, Franke C. Kinetics of AMPA-type receptor channels in rat caudate-putame neurones show a wide range of desensitization but distinct recovery characteristics.  Eur J Neurosci. 1998;  10 664-672
• 13 Koike M, Tsukada S, Tsuzuki K, Kijima H, Ozawa S. Regulation of kinetic properties of GluR2 AMPA receptor channels by alternative splicing.  J Neurosci. 2000;  20 2166-2174
• 14 Krampfl K, Schlesinger F, Zörner A, Kappler M, Dengler R, Bufler J. Control of kinetic properties of GluR2 flop AMPA-type channels: impact of R/G nuclear editing.  Eur J Neurosci. 2002;  15 51-62
• 15 Schlesinger F, Tammena D, Krampfl K, Bufler J. Desensitization and resensitization are independently regulated in human GluR subunit coassemblies.  Synapse. 2005;  55 176-182
• 16 Bufler J, Wilhelm R, Parnas H, Franke C, Dudel J. Open channel and competitive block of embryonic form of the nicotinic receptor of mouse myotubes by (+)-tubocurarine.  J Physiol. 1996;  495 83-95
• 17 Honore T, Davis S N, Drejer J, Fletcher E J, Jacobsen P, Lodge D, Nielsen F E. Quinoxalidiones: potent non-NMDA glutamate receptor antagonists.  Science. 1988;  241 701-703
• 18 Sheardown M J, Nielsen E O, Hansen A J, Jacobsen P, Honore T. 2,3-Dihydroxy-6-nitro-7-sulfomoyl-benzo(F)-quinoxaline: a neuroprotectant for cerebral ischemia.  Science. 1990;  247 571-574
• 19 Okda M, Kohara H, Yamaguchi T. Characterization of YM90K, a selective and potent antagonist of AMPA receptors, in rat cortical mRNA-injected oocytes.  Eur J Pharmacol. 1996;  309 299-306
• 20 Bowie D, Lange G D, Mayer M L. Activity-dependent modulation of glutamate receptors by polyamines.  J Neurosci. 1998;  18 8175-8185
• 21 Canton T, Böhme G A, Boireau G A, Bordier A, Mignani S, Jimonet P, Jahn G, Alavijeh M, Stygall J, Roberts S, Brealey C, Vuilhorgne M, Debono M-W, Guern S Le, Laville M, Briet D, Roux M, Stutzmann J-M, Pratt J. RPR119990, a novel α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid antagonist: synthesis, pharmacological properties, and activity in an animal model of amyotrophic lateral sclerosis.  J Pharmacol Exp Ther. 2001;  299 314-322
• 22 Mignani S, Bohme G A, Birraux G, Boireau A, Jimonet P, Damour D, Genevois-Borella A, Debono M-W, Pratt J, Vuilhorgne M, Wahl F, Stutzmann J-M. 9-Carboxymethyl-5H,10H-imidazo[1,2-a]indeno[1,2-e]pyrazin-4-one-2carbocylic acid (RPR117824): selective anticonvulsive and neuroprotective AMPA antagonist.  Bioorgan Medic Chem. 2002;  10 1627-1637
• 23 Hamill O P, Marty A, Neher E, Sakmann B, Sigworth F J. Improved patch-clamp techniques for high resolution current recordings from cells and cell-free patches.  Pflügers Arch. 1981;  391 85-100
• 24 Franke C, Hatt H, Dudel J. Liquid filament switch for ultra-fast exchanges of solutions at excised patches of synaptic membrane of crayfish muscle.  Neurosci Lett. 1987;  77 199-204
• 25 Krampfl K, Schlesinger F, Cordes A L, Bufler J. Molecular analysis of the interaction of the pyrazine derivatives RPR119990 and RPR117824 with human AMPA-type glutamate receptor channels.  Neuropharm. 2006;  50 479-490
• 26 Schlesinger F, Tammena D, Krampfl K, Bufler J. Two mechanisms of action of the adamantane derivative IEM-1460 at human AMPA-type glutamate receptors.  Br J Pharmacol. 2005;  145 656-663